Journal of Plant Growth Regulation

, Volume 35, Issue 3, pp 744–754 | Cite as

Comparative Proteomic Analysis Reveals Nitrogen Fertilizer Increases Spikelet Number per Panicle in Rice by Repressing Protein Degradation and 14-3-3 Proteins

  • Chengqiang Ding
  • Yan Wang
  • Zhongyuan Chang
  • Siliang You
  • Zhenghui Liu
  • Shaohua Wang
  • Yanfeng Ding


The spikelet number per panicle is established in the early stages of panicle development. Nitrogen fertilizer application before panicle initiation is known to increase spikelet number, which is one of the most important traits in rice productivity determination. However, the basic proteomic mechanism remains poorly understood. The present study shows that nitrogen fertilizer significantly increased spikelet number and grain yield in rice. Proteomic variations were further analyzed in young panicles at the secondary panicle branch initiation and spikelet meristem initiation under nitrogen fertilizer treatment. Proteomic analysis identified 63 proteins with significant differential accumulation in young panicles under nitrogen fertilizer treatment. Proteolysis represents the largest functional category, which suggests that protein degradation is an important pathway in the response to nitrogen fertilizer. Importantly, nitrogen fertilizer significantly reduced 14-3-3 proteins, which interact with key enzymes associated with carbon and nitrogen metabolism, and the rice FT homologue Hd3a. Real-time PCR revealed that Hd3a signaling is also repressed by nitrogen fertilizer in leaves. This study contributes to a better understanding of the regulation of nitrogen fertilizers in the flowering pathway leading to panicle development. The identification of novel genes provides new insight into the profound impacts of nitrogen fertilizer on panicle development in rice.


14-3-3 proteins, nitrogen fertilizer Rice Spikelet number Protein degradation 



This work was supported by the National Natural Science Foundation of China (Grant Numbers 31401324; 31401324) and the National High Technology Research and Development Program of China (863 Program, Grant Number 2014AA10A605-1).

Author contributions

Chengqiang Ding, Shaohua Wang, and Yanfeng Ding designed research; Chengqiang Ding performed research and analyzed data; and Chengqiang Ding, Yan Wang, and Siliang You wrote the paper.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

344_2016_9579_MOESM1_ESM.pdf (152 kb)
Supplementary material 1 (PDF 152 kb)
344_2016_9579_MOESM2_ESM.pdf (38 kb)
Supplementary material 2 (PDF 38 kb)
344_2016_9579_MOESM3_ESM.pdf (23 kb)
Supplementary material 3 (PDF 23 kb)
344_2016_9579_MOESM4_ESM.pdf (65 kb)
Supplementary material 4 (PDF 64 kb)
344_2016_9579_MOESM5_ESM.xlsx (86 kb)
Supplementary material 5 (XLSX 86 kb) Fragment sequence and the individual score of each protein in this paper


  1. Alsterfjord M, Sehnke PC, Arkell A, Larsson H, Svennelid F, Rosenquist M, Ferl RJ, Sommarin M, Larsson C (2004) Plasma membrane H(+)-ATPase and 14-3-3 isoforms of Arabidopsis leaves: evidence for isoform specificity in the 14-3-3/H(+)-ATPase interaction. Plant Cell Physiol 45:1202–1210CrossRefGoogle Scholar
  2. Bornke F (2005) The variable C-terminus of 14-3-3 proteins mediates isoform-specific interaction with sucrose-phosphate synthase in the yeast two-hybrid system. J Plant Physiol 162:161–168CrossRefGoogle Scholar
  3. Chen X, Cui Z, Fan M, Vitousek P, Zhao M, Ma W, Wang Z, Zhang W, Yan X, Yang J, Deng X, Gao Q, Zhang Q, Guo S, Ren J, Li S, Ye Y, Wang Z, Huang J, Tang Q, Sun Y, Peng X, Zhang J, He M, Zhu Y, Xue J, Wang G, Wu L, An N, Wu L, Ma L, Zhang W, Zhang F (2014) Producing more grain with lower environmental costs. Nature 514:486–489CrossRefPubMedGoogle Scholar
  4. Chi F, Yang P, Han F, Jing Y, Shen S (2010) Proteomic analysis of rice seedlings infected by Sinorhizobium meliloti 1021. Proteomics 10:1861–1874CrossRefPubMedGoogle Scholar
  5. Diaz C, Kusano M, Sulpice R, Araki M, Redestig H, Saito K, Stitt M, Shin R (2011) Determining novel functions of Arabidopsis 14-3-3 proteins in central metabolic processes. BMC Syst Biol 5:192CrossRefPubMedPubMedCentralGoogle Scholar
  6. Ding C, You J, Liu Z, Rehmani MIA, Wang S, Li G, Wang Q, Ding Y (2011) Proteomic analysis of low nitrogen stress-responsive proteins in roots of rice. Plant Mol Biol Rep 29:618–625CrossRefGoogle Scholar
  7. Ding C, You J, Wang S, Liu Z, Li G, Wang Q, Ding Y (2012) A proteomic approach to analyze nitrogen- and cytokinin-responsive proteins in rice roots. Mol Biol Rep 39:1617–1626CrossRefPubMedGoogle Scholar
  8. Ding C, You J, Chen L, Wang S, Ding Y (2014) Nitrogen fertilizer increases spikelet number per panicle by enhancing cytokinin synthesis in rice. Plant Cell Rep 33:363–371CrossRefPubMedGoogle Scholar
  9. Furutani I, Sukegawa S, Kyozuka J (2006) Genome-wide analysis of spatial and temporal gene expression in rice panicle development. Plant J 46:503–511CrossRefPubMedGoogle Scholar
  10. Gampala SS, Kim TW, He JX, Tang W, Deng Z, Bai MY, Guan S, Lalonde S, Sun Y, Gendron JM, Chen H, Shibagaki N, Ferl RJ, Ehrhardt D, Chong K, Burlingame AL, Wang ZY (2007) An essential role for 14-3-3 proteins in brassinosteroid signal transduction in Arabidopsis. Dev Cell 13:177–189CrossRefPubMedPubMedCentralGoogle Scholar
  11. Guo QF, Zhang J, Gao Q, Xing SC, Li F, Wang W (2008) Drought tolerance through overexpression of monoubiquitin in transgenic tobacco. J Plant Physiol 165:1745–1755CrossRefPubMedGoogle Scholar
  12. Hakeem KR, Chandna R, Ahmad A, Qureshi MI, Iqbal M (2012) Proteomic analysis for low and high nitrogen-responsive proteins in the leaves of rice genotypes grown at three nitrogen levels. Appl Biochem Biotechnol 168:834–850CrossRefPubMedGoogle Scholar
  13. Ju XT, Xing GX, Chen XP, Zhang SL, Zhang LJ, Liu XJ, Cui ZL, Yin B, Christie P, Zhu ZL, Zhang FS (2009) Reducing environmental risk by improving N management in intensive Chinese agricultural systems. Proc Natl Acad Sci USA 106:3041–3046CrossRefPubMedPubMedCentralGoogle Scholar
  14. Kamada-Nobusada T, Makita N, Kojima M, Sakakibara H (2013) Nitrogen-dependent regulation of De Novo cytokinin biosynthesis in rice: the role of glutamine metabolism as an additional signal. Plant Cell Physiol 54:1881–1893CrossRefPubMedPubMedCentralGoogle Scholar
  15. Komiya R, Ikegami A, Tamaki S, Yokoi S, Shimamoto K (2008) Hd3a and RFT1 are essential for flowering in rice. Development 135:767–774CrossRefPubMedGoogle Scholar
  16. Kurakawa T, Ueda N, Maekawa M, Kobayashi K, Kojima M, Nagato Y, Sakakibara H, Kyozuka J (2007) Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445:652–655CrossRefPubMedGoogle Scholar
  17. Li M, Tang D, Wang K, Wu X, Lu L, Yu H, Gu M, Yan C, Cheng Z (2011) Mutations in the F-box gene LARGER PANICLE improve the panicle architecture and enhance the grain yield in rice. Plant Biotechnol J 9:1002–1013CrossRefPubMedGoogle Scholar
  18. Lian X, Wang S, Zhang J, Feng Q, Zhang L, Fan D, Li X, Yuan D, Han B, Zhang Q (2006) Expression profiles of 10,422 genes at early stage of low nitrogen stress in rice assayed using a cDNA microarray. Plant Mol Biol 60:617–631CrossRefPubMedGoogle Scholar
  19. Liao C, Peng Y, Ma W, Liu R, Li C, Li X (2012) Proteomic analysis revealed nitrogen-mediated metabolic, developmental, and hormonal regulation of maize (Zea mays L.) ear growth. J Exp Bot 63:5275–5288CrossRefPubMedPubMedCentralGoogle Scholar
  20. Lyzenga WJ, Stone SL (2012) Abiotic stress tolerance mediated by protein ubiquitination. J Exp Bot 63:599–616CrossRefPubMedGoogle Scholar
  21. Makino A (2011) Photosynthesis, grain yield, and nitrogen utilization in rice and wheat. Plant Physiol 155:125–129CrossRefPubMedGoogle Scholar
  22. Moorhead G, Douglas P, Morrice N, Scarabel M, Aitken A, MacKintosh C (1996) Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins and activated by fusicoccin. Curr Biol 6:1104–1113CrossRefPubMedGoogle Scholar
  23. Muthayya S, Sugimoto JD, Montgomery S, Maberly GF (2014) An overview of global rice production, supply, trade, and consumption. Ann N Y Acad Sci 1324:7–14CrossRefPubMedGoogle Scholar
  24. Peng M, Hannam C, Gu H, Bi YM, Rothstein SJ (2007) A mutation in NLA, which encodes a RING-type ubiquitin ligase, disrupts the adaptability of Arabidopsis to nitrogen limitation. Plant J 50:320–337CrossRefPubMedGoogle Scholar
  25. Purwestri YA, Ogaki Y, Tamaki S, Tsuji H, Shimamoto K (2009) The 14-3-3 protein GF14c acts as a negative regulator of flowering in rice by interacting with the florigen Hd3a. Plant Cell Physiol 50:429–438CrossRefPubMedGoogle Scholar
  26. Riedel J, Tischner R, Mack G (2001) The chloroplastic glutamine synthetase (GS-2) of tobacco is phosphorylated and associated with 14-3-3 proteins inside the chloroplast. Planta 213:396–401CrossRefPubMedGoogle Scholar
  27. Rieu I, Powers SJ (2009) Real-time quantitative RT-PCR: design, calculations, and statistics. Plant Cell 21:1031–1033CrossRefPubMedPubMedCentralGoogle Scholar
  28. Sato T, Maekawa S, Yasuda S, Domeki Y, Sueyoshi K, Fujiwara M, Fukao Y, Goto DB, Yamaguchi J (2011a) Identification of 14-3-3 proteins as a target of ATL31 ubiquitin ligase, a regulator of the C/N response in Arabidopsis. Plant J 68:137–146CrossRefPubMedGoogle Scholar
  29. Sato T, Maekawa S, Yasuda S, Yamaguchi J (2011b) Carbon and nitrogen metabolism regulated by the ubiquitin-proteasome system. Plant Signal Behav 6:1465–1468CrossRefPubMedPubMedCentralGoogle Scholar
  30. Sehnke PC, Chung HJ, Wu K, Ferl RJ (2001) Regulation of starch accumulation by granule-associated plant 14-3-3 proteins. Proc Natl Acad Sci USA 98:765–770CrossRefPubMedPubMedCentralGoogle Scholar
  31. Shabek N, Zheng N (2014) Plant ubiquitin ligases as signaling hubs. Nat Struct Mol Biol 21:293–296CrossRefPubMedGoogle Scholar
  32. Smalle J, Kurepa J, Yang P, Emborg TJ, Babiychuk E, Kushnir S, Vierstra RD (2003) The pleiotropic role of the 26S proteasome subunit RPN10 in Arabidopsis growth and development supports a substrate-specific function in abscisic acid signaling. Plant Cell 15:965–980CrossRefPubMedPubMedCentralGoogle Scholar
  33. Takai R, Matsuda N, Nakano A, Hasegawa K, Akimoto C, Shibuya N, Minami E (2002) EL5, a rice N-acetylchitooligosaccharide elicitor-responsive RING-H2 finger protein, is a ubiquitin ligase which functions in vitro in co-operation with an elicitor-responsive ubiquitin-conjugating enzyme, OsUBC5b. Plant J 30:447–455CrossRefPubMedGoogle Scholar
  34. Tamaki S, Matsuo S, Wong HL, Yokoi S, Shimamoto K (2007) Hd3a protein is a mobile flowering signal in rice. Science 316:1033–1036CrossRefPubMedGoogle Scholar
  35. Taoka K, Ohki I, Tsuji H, Furuita K, Hayashi K, Yanase T, Yamaguchi M, Nakashima C, Purwestri YA, Tamaki S, Ogaki Y, Shimada C, Nakagawa A, Kojima C, Shimamoto K (2011) 14-3-3 proteins act as intracellular receptors for rice Hd3a florigen. Nature 476:332–335CrossRefPubMedGoogle Scholar
  36. Taoka K, Ohki I, Tsuji H, Kojima C, Shimamoto K (2013) Structure and function of florigen and the receptor complex. Trends Plant Sci 18:287–294CrossRefPubMedGoogle Scholar
  37. Tian FX, Gong JF, Zhang J, Feng YA, Wang GK, Guo QF, Wang W (2014) Overexpression of monoubiquitin improves photosynthesis in transgenic tobacco plants following high temperature stress. Plant Sci 226:92–100CrossRefPubMedGoogle Scholar
  38. Wang X, Li Y, Fang G, Zhao Q, Zeng Q, Li X, Gong H, Li Y (2014) Nitrite promotes the growth and decreases the lignin content of indica rice Calli: a comprehensive transcriptome analysis of nitrite-responsive genes during in vitro culture of rice. PLoS One 9:e95105CrossRefPubMedPubMedCentralGoogle Scholar
  39. Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ (2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21:3666–3672CrossRefPubMedGoogle Scholar
  40. Yao Y, Du Y, Jiang L, Liu JY (2007) Interaction between ACC synthase 1 and 14-3-3 proteins in rice: a new insight. Biochemistry (Mosc) 72:1003–1007CrossRefGoogle Scholar
  41. Yasuda S, Sato T, Maekawa S, Aoyama S, Fukao Y, Yamaguchi J (2014) Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 is critical for plant C/N-nutrient response and controls the stability of 14-3-3 proteins. J Biol Chem 289(22):15179–15193CrossRefPubMedPubMedCentralGoogle Scholar
  42. Yoshida H, Horie T, Shiraiwa T (2006) A model explaining genotypic and environmental variation of rice spikelet number per unit area measured by cross-locational experiments in Asia. Field Crops Res 97:337–343CrossRefGoogle Scholar
  43. Zhang J, Guo QF, Feng YN, Li F, Gong JF, Fan ZY, Wang W (2012) Manipulation of monoubiquitin improves salt tolerance in transgenic tobacco. Plant Biol 14:315–324CrossRefPubMedGoogle Scholar
  44. Zheng YS, Guo JX, Zhang JP, Gao AN, Yang XM, Li XQ, Liu WH, Li LH (2013) A proteomic study of spike development inhibition in bread wheat. Proteomics 13:2622–2637CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Chengqiang Ding
    • 1
  • Yan Wang
    • 1
  • Zhongyuan Chang
    • 1
  • Siliang You
    • 1
  • Zhenghui Liu
    • 1
  • Shaohua Wang
    • 1
  • Yanfeng Ding
    • 1
  1. 1.College of AgronomyNanjing Agricultural UniversityNanjingPeople’s Republic of China

Personalised recommendations